Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing (original) (raw)
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The EMBO Journal, 2002
Posttranscriptional gene silencing (PTGS) processes double-stranded (ds) RNAs into 21±25 nucleotide (nt) RNA fragments that direct ribonucleases to target cognate mRNAs. In higher plants, PTGS also generates mobile signals conferring sequence-speci®c silencing in distant organs. Since PTGS acts as an antiviral system in plants, successful virus infection requires evasion or suppression of gene silencing. Here we report that the 19 kDa protein (p19) of tombusviruses is a potent silencing suppressor that prevents the spread of mobile silencing signal. In vitro, p19 binds PTGS-generated, 21±25 nt dsRNAs and 21-nt synthetic dsRNAs with 2-nt 3¢ overhanging end(s), while it barely interacts with single-stranded (ss) RNAs, long dsRNAs or blunt-ended 21-nt dsRNAs. We propose that p19 mediates silencing suppression by sequestering the PTGS-generated 21±25 nt dsRNAs, thus depleting the speci®city determinants of PTGS effector complexes. Moreover, the observation that p19-expressing transgenic plants show altered leaf morphology might indicate that the p19-targeted PTGS pathway is also important in the regulation of plant development.
Journal of Virology, 2005
RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus Tombusvirus. We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus-and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.
Identification of an RNA Silencing Suppressor from a Plant Double-Stranded RNA Virus
Journal of Virology, 2005
RNA silencing is a mechanism which higher plants and animals have evolved to defend against viral infection in addition to regulation of gene expression for growth and development. As a counterdefense, many plant and some animal viruses studied to date encode RNA silencing suppressors (RSS) that interfere with various steps of the silencing pathway. In this study, we report the first identification of an RSS from a plant double-stranded RNA (dsRNA) virus. Pns10, encoded by S10 of Rice dwarf phytoreovirus RDV), exhibited RSS activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c carrying GFP. The other gene segments of the RDV genome did not have such a function. Pns10 suppressed local and systemic silencing induced by sense RNA but did not interfere with local and systemic silencing induced by dsRNA. Expression of Pns10 also increased the expression of -glucuronidase in transient assays and enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, our results establish Pns10 as an RSS encoded by a plant dsRNA virus and further suggest that Pns10 targets an upstream step of dsRNA formation in the RNA silencing pathway.
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2009
Small RNAs, including small interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi-associated interfering RNAs (piRNAs), are powerful gene expression regulators. This RNA-mediated regulation results in sequencespecific inhibition of gene expression by translational repression and/or mRNA degradation. siRNAs and miRNAs are generated by RNase III enzymes and subsequently loaded into Argonaute protein, a key component of the RNA induced silencing complex (RISC), to form the core of the RNA silencing machinery. RNA silencing acts as an ancient cell defense system against molecular parasites, such as transgenes, viruses and transposons. RNA silencing also plays an important role in the control of development. In plants, RNA silencing serves as a potent antiviral defense system. In response, many viruses have developed strategies to suppress RNA silencing. The striking sequence diversity among viral suppressors suggests that different viral suppressors could target different components of the RNA silencing machinery at different steps in different suppressing modes. Significant progresses have been made in this field for the past 5 years on the basis of structural information derived from RNase III family proteins, Dicer fragments and homologs, Argonaute homologs and viral suppressors. In this paper, we will review the current progress on the understanding of molecular mechanisms of RNA silencing; highlight the structural principles determining the protein-RNA recognition events along the RNA silencing pathways and the suppression mechanisms displayed by viral suppressors.
Approaches to Plant Stress and their Management, 2013
Small RNA molecules play a crucial regulatory role in maintaining genome stability as well as developmental regulations through a set of complex and partially overlapping pathways in a wide range of eukaryotic organisms. Active in both cytoplasm and nucleus, RNA interference regulates eukaryotic gene expression through transcriptional repression by epigenetic modification and interaction with transcription machinery. Small interfering RNAs (siRNAs/miRNAs) of 21-24 nucleotides constitute the innate defence arm against a variety of pathogens, especially viruses. Plant viruses with either DNA or RNA genomes are subjected to small RNA-directed RNA degradation. Additionally, DNA viruses are subjected to another line of defence through 'RNA-directed DNA methylations' (RdDM). On the other hand, viral-encoded proteins, called silencing suppressors (VSRs), are known to counter the defence machinery, and therefore the virus can evade the host surveillance system. Some plant viruses additionally adopt certain strategies like acquiring silencing resistant structures (some RNA virus) to evade the RNA silencing machinery and thereby shaping the viral as well as the host genome. Recently, it has been reported that particular viral proteins and viral siRNAs contribute directly to pathogenicity by interacting with certain host proteins or RNAs. Transcriptional regulation of host gene by small RNA of viral origin plays important role in pathogenesis and symptom development. Small regulatory RNAs of cellular rather than pathogen origin have also been found to play a broad role in improving the basal defence in the case of plant-virus interaction. This chapter provides key insights into the complex intricate machinery of diverse RNA silencing mechanisms, describes various evolutionary diverse strategies of viral
Viral Suppressors of RNA Silencing in Plants
In eukaryotes, small RNAs play a crucial regulatory role in many processes including development, maintenance of genome stability and antiviral responses. These different but overlapping RNA-guided pathways are collectively termed 'RNA silencing'. In plants, RNA silencing serves as a major line of antiviral defense that is induced by, and targeted against viruses. As a counter-defensive strategy, viruses have evolved to encode suppressor proteins that inhibit various stages of the silencing process. These suppressors are diverse in sequence and structure and appear to be encoded by virtually any type of plant viruses. This review focuses on the novel methods of suppressor screening and revealing the characteristics of RNA silencing suppressors. We have also discussed the mechanism of suppression activity, which principally operate by modifying the accumulation, activity, and/or transmission of siRNAs through either direct interaction with the RNA species or components of the RNA silencing machinery. Finally, the biotechnological applications of silencing suppression have been considered.
Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome
Proceedings of the National Academy of Sciences, 2004
virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the Ϸ20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP-or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host.
RNA, 2011
RNA silencing mediated by siRNAs plays an important role as an anti-viral defense mechanism in plants and other eukaryotic organisms, which is usually counteracted by viral RNA silencing suppressors (RSSs). The ipomovirus Cucumber vein yellowing virus (CVYV) lacks the typical RSS of members of the family Potyviridae, HCPro, which is replaced by an unrelated RSS, P1b. CVYV P1b resembles potyviral HCPro in forming complexes with synthetic siRNAs in vitro. Electrophoretic mobility shift assays showed that P1b, like potyviral HCPro, interacts with double-stranded siRNAs, but is not able to bind single-stranded small RNAs or small DNAs. These assays also showed a preference of CVYV P1b for binding to 21-nt siRNAs, a feature also reported for HCPro. However, these two potyvirid RSSs differ in their requirements of 2-nucleotide (nt) 39 overhangs and 59 terminal phosphoryl groups for siRNA binding. Copurification assays confirmed in vivo P1b-siRNA interactions. We have demonstrated by deep sequencing of small RNA populations interacting in vivo with CVYV P1b that the size preference of P1b for small RNAs of 21 nt also takes place in the plant, and that expression of this RSS causes drastic changes in the endogenous small RNA populations. In addition, a site-directed mutagenesis analysis strongly supported the assumption that P1b-siRNA binding is decisive for the anti-silencing activity of P1b and localized a basic domain involved in the siRNA-binding activity of this protein.
RNA silencing suppressors encoded by viruses of the family Tombusviridae
Plant Biotechnology, 2005
RNA silencing is small RNA-guided sequence-specific gene inactivation mechanism in eukaryote that is involved in diverse biological phenomena (e.g. development, heterochromatin formation, and defense against molecular parasites such as transposons and viruses) (reviewed by Baulcombe 2004; Vastenhouw and Plasterk 2004; Voinnet 2005). RNA silencing is induced by double-stranded RNAs (dsRNAs) that are the precursors of small RNAs. In this review, we do not address heterochromatin formation or repeat-associated small-interfering RNA (rasiRNA), but we refer to other reviews of these topics (Sontheimer and Carthew 2005; Wassenegger 2005). RNA silencing pathways in plants other than those involved in heterochromatin formation and in rasiRNA are illustrated in Figure 1. In plants, the small RNAs involved in RNA silencing are 21-24 nucleotides (nt) (Hamilton et al. 2002) and are classified into three distinct RNA groups depending on their biogenesis. First, small-interfering RNA (siRNA) is processed from dsRNAs that are mainly derived from molecular parasites, including transposons, viruses, transgenes, and exogeneously supplied dsRNAs (Hamilton and Baulcombe 1999). siRNA is involved in defense against molecular parasites. siRNA-mediated gene inactivation is now widely used in molecular biology. Second, microRNA (miRNA) (reviewed by Bartel 2004) is processed from short endogenous RNA hairpins that are derived from miRNA precursors transcribed by RNA polymerase II. miRNA regulates the expression of genes from mRNA with nucleotide sequences complementary to the miRNA by mRNA cleavage or translation repression, to control diverse developmental processes. Interestingly, miRNA is involved in antiviral defenses in animals (Lecellier et al. 2005). It is unclear whether miRNA is involved in antiviral defenses in plants. Third, transacting siRNA (ta-siRNA) is a recently identified endogenous siRNA (Peragine et al. 2004; Vazquez et al. 2004). The generation of ta-siRNA requires miRNA-mediated mRNA cleavage and subsequent dsRNA synthesis by an RNA-dependent RNA polymerase (RdRP), RDR6 (Allen et al. 2005). ta-siRNA regulates gene expression in a way similar to that of miRNA. The key molecules in RNA silencing in plants are the Dicer-like enzymes (DCLs), Argonaute proteins (AGOs), and RdRPs (Reviewed by Baulcombe 2004). The Arabidopsis thaliana genome encodes four DCLs, ten AGOs, and six RdRPs. DCL is an RNase III enzyme that